Biophysical studies of peptides with functions in biotechnology and biology

iii To My Dear Family iv v Abstract My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles). The resorufin-based arsenical hairpin binder (ReAsH) bound to a short pep-tide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to a type II β-turn was determined by NMR structure calculations (Paper I). Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV. The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency. Endosomal escape is a crucial challenge for the CPP applications. We mod-eled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs. Dynorphin A (Dyn A) peptide mutations are associated with neurodegenera-tive disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects. ix Abbreviations As Arsenic BR Bacteriorhodopsin CD Circular dichroism CPP Cell-penetrating peptide CQ Chloroquine Cy5 Cyanine 5 DLS Dynamic light scattering DQF COSY Double quantum filtered-correlation spectroscopy Dyn Dynorphin EDT Ethanedithiol FACS Fluorescence-assisted cell sorting FITC Fluorescein isothiocyanate FlAsH Fluorescein-based arsenical hairpin binder FRET Fluorescence resonance energy transfer GFP Green fluorescent protein GUV Giant unilamellar vesicle HIV Human immunodeficiency virus HS Heparan sulfate HsapBKp Paddle motif from Homo sapience large-conductance calcium activated potassium channel (BK) HSQC Heteronuclear single quantum coherence IC Internal conversion ISC Intersystem crossing KvAPp Paddle motif from voltage-gated potassium-channel (Aeropy-rum pernix) LUV Large unilamellar vesicle MAP Model amphipathic peptide MLV Multilamellar …